Quantifying exposure to stress

ABSTRACT

The present invention provides an in vitro method for quantifying exposure to psychological stress which relies on measuring the retained ability of neutrophils, preferably neutrophils in a whole blood sample, to exhibit challenge-induced superoxide anion production. Using such methodology, coping capacity of individuals for particular psychological stressors may be assessed.

The present invention relates to in vitro methods of quantifyingexposure to psychological stress in an individual, which may be a humanor non-human animal. More particularly, the present invention provides amethod of quantifying exposure to psychological stress which relies onmeasuring the retained ability of neutrophils, preferably neutrophils ina whole blood sample, to exhibit challenge-induced superoxide anionproduction, i.e. produce a “respiratory burst” in response to in vitroactivation. Such methodology may also be applied to screen forstress-relieving drugs.

BACKGROUND TO THE INVENTION

Objective, quantitative and practicable measures of psychological stressare pivotal to studies in many branches of vertebrate biology, includingwildlife conservation and management, and are also relevant to thefarming industry, animal welfare and veterinary services. Techniques forquantifying levels of human stress are important from a medicalperspective, but are also of interest for occupational safety andhealth. An estimated 1 in 10 people in the UK suffer from work relatedstress (Health & Safety Executive Information sheet: 1/01/EMSU updatedFebruary 2002) at a cost of £3.7 billion to society. Stress-relatedconditions or effects include immune disorders, cardiovascular disease,muscoskeletal and psychological disorders, workplace injury and ulcers.

Various methods have previously been applied in an attempt to assessstress responses, including the perceived stress questionnaire (Cohen etal., J. Health & Social Behaviour (1983) 24, 385-396), measurement ofcortisol and hormonal levels in blood, urine, saliva and faeces (Beerdaet al., Horm. Behav. (1996) 30, 272-279), haematological values (e.g.Millspaugh et al., Can. Field-Nat. (2000) 114, 196-200) and behaviouralobservations (reviewed by Rushen in The Biology of Animal Stress BasicPrinciples And Implications For Animal Welfare (2000) ed. Moberg &Mench, Wallingford:CABI Publishing). However, these approaches havevarious drawbacks, and may be inappropriate to use in certainsituations. The perceived stress questionnaire is subjective, and socannot provide an objective measure of stress. Furthermore, questionsmay not always be answered honestly, for example due to culturalreasons, and this measure of stress cannot be used for testing animalsor birds. Measurements of cortisol and hormonal levels in, for example,blood and urine, are often used to assess stress in animals, but thesemeasures are not ideal since their levels change throughout the year.There are no absolute terms of reference, and this type of testing istime consuming. Behavioural observations are also often subjective anddifficult to quantify.

More recently, it was shown that exposure of human volunteers to a shortterm mental stressor leads to increased superoxide anion productionbeing observable in neutrophils present in peripheral blood samples(Ellard et al., Inter. J. Psychophys. (2001) 41, 93-100). It has nowbeen found that exposure of both animals and humans to psychologicalstress can be rapidly and readily quantified by relying on measurementof the retained capacity of neutrophils in peripheral blood samples toproduce superoxide anions in response to a challenge by phorbolmyristate acetate (PMA), a known chemical-inducer for activatingneutrophils (Hu et al., Cell Signal (1999) 11, 355-360). By this means,for the first time, it has proved possible to obtain a quantitativemeasurement of copying capacity of animals and humans for known orsuspected psychological stressors.

SUMMARY OF THE INVENTION

The method of the invention for quantifying stress is based on directmeasures of a cellular response which forms part of the armoury of theimmune system. It relies on the ability of individuals to mount achallenge-induced immune response after a potentially stressful event.Each individual's capacity to respond to immune challenge is comparedwith their own baseline level of immune system activity. After exposureto a known or suspected psychological stressor, superoxide anionproduction in neutrophils is stimulated iii vitro, and the capacity ofneutrophils to produce further superoxide is measured, in effectdetermining the extent to which superoxide production has beendiminished by the known or suspected stressful event. The ability ofneutrophils to respond to such in vitro challenge after a stressfulevent is defined as the individual's coping capacity. Individuals with ahigher coping capacity have a greater potential superoxide productionand, physiologically, are better able to cope with bacterial challengeafter stress. Therefore coping capacity is an in vitro assessment of theindividual's current physiological status.

In its broadest aspect, the present invention therefore provides amethod for determining whether an individual, which may be a mammal,including a human, or a bird, is experiencing changed physiologicalstatus arising from a psychological stressor, the method comprising:

-   -   (a) contacting a test sample comprising neutrophils obtained        from said individual with an inducer capable of stimulating        superoxide production in neutrophils under conditions suitable        for such stimulation;    -   (b) determining the increase in superoxide production above        basal in said test sample after a time period when neutrophils        of the same species in a control sample, which are free or        substantially free of stress-induced activation or at least        derived from one or more individuals exposed to the same regime        minis a factor to be tested as a psychological stressor, will        exhibit superoxide production under the same in vitro        conditions; and    -   (c) comparing the increase in superoxide production above basal        observed in said test sample with the increase in superoxide        production above basal observed in a control sample as defined        in (b) above under the same conditions;        wherein lower superoxide production in said test sample is        indicative of the effect of a psychological stressor on the        individual's physiological status. If need be or desired, the        measured superoxide production above basal for each sample may        be corrected by reference to the number of leucocytes or        neutrophils in the sample (although a range of leucocytes can        produce a respiratory burst, neutrophils are responsible for the        majority of superoxide production). As indicated above, residual        capacity of leucocytes, or more particularly neutrophils, above        basal for in vitro-induced superoxide induction can be termed        coping capacity.

The control neutrophils may not be entirely free of stress-inducedactivation but will necessarily be obtained from the same individual oran individual of the same species prior to exposure of the individual toa suspected or known psychological stressor selected for study. Forexample, Example 1 illustrates application of such methodology tobadgers where the additional stress-inducing effect of transport wasquantified compared to mere badger capture based on comparing theresidual capacity for challenge-induced activation of neutrophils fromtransported badgers with such residual capacity of neutrophils fromcaptured but not transported badgers (the controls). Example 2 furtherillustrates application of the invention for quantifying thestress-inducing effect of handling on wild mammals, e.g. wild mammals asmay be found in the UK countryside. Indeed, the invention has wideapplication in the fields of wildlife conservation, veterinary scienceand animal husbandry for improving animal well-being, as well asapplications to humans as further discussed below.

The samples employed in a method of the invention may be mostconveniently whole peripheral blood samples and hence such a methodprovides rapid results and can be utilised in the field or laboratory.Such a method can be used to elucidate psychological stressors or todetermine whether an individual is suffering from recent or continuedexposure to a psychological stressor. As indicated above, mostimportantly, for the first time the present invention provides a meansof quantifying the ability of individuals to cope with known orsuspected psychological stressors.

Hence, in a preferred embodiment, there is provided a method fordetermining the coping capacity of an individual for exposure to apsychological stressor, wherein prior to step (a) in a method as definedabove said individual is exposed to said psychological stressor for atime period whereby neutrophils in an individual of the same species whois susceptible to stress induced by said stressor will exhibit increasedsuperoxide production and wherein the degree of further in vitro inducedsuperoxide production in said test sample above basal determined in step(c) is a measure of coping capacity.

In a variation of the above-described methods of the invention, thecontrol sample is substituted by a second test sample comprisingneutrophils derived from a second individual, the tests samples beingtaken from individuals at the same time point before, during or aftersubjection to different regimes which it is desired to compare asstressors, e.g. before, during or after different protocols for medicaltreatment. In this case, superoxide production above basal for eachsample will be corrected with reference to the white cell count orneutrophil count. This gives a measure of coping capacity standardisedto allow for differences in the number of white cells in the peripheralblood of the selected individuals and thereby enables quantitativecomparison of the stress-inducing effect of the two regimes. Suchcomparison may be a carried out at more than one time point andgenerally samples from a number of individuals subjected to the twodifferent regimes will be tested. Application of such methodology tocompare perceived stress and measured decrease of leucocyte copingcapacity associated with elective and acute cardio-pulmonary bypasssurgery is detailed in Example 6. Such methodology of the invention hasmany applications for optimising procedures to minimise psychologicalstress and provides a highly convenient quantitative method forevaluating proposals for alleviating psychological stress associatedwith procedures known to be stressful to human, non-human mammals andbirds.

BRIEF DESCRIPTION OF THE DRAWINGS

Example 1 illustrates use of the methodology of the invention to testthe prediction that badgers (Meles meles) have a lower coping capacitywhen they are subjected to trapping and then transport than when theyare trapped but do not experience the additional stress of transportwith reference to the following figures:

FIG. 1 shows coping capacity measured as relative light units (solidlines, mean±SE) in 8 badgers after transport (square), and 8 badgerswithout transport (circle) as determined by chemiluminescencemeasurement of superoxide production in whole blood after challenge withPMA. Dashed lines represent basal leucocyte activity.

FIG. 2 shows coping capacity calculated as relative light units (RLU)per 10⁹ neutrophils 1⁻¹ Solid lines represent coping capacity (mean±SE)in badgers after transport (square), and without transport (circle).Dashed lines represent basal activity.

Example 2 illustrates use of the invention to quantify stress induced insmall wild animals (wood mice and bank voles) by handling with referenceto the following figure:

FIG. 3 shows coping capacity measured in relative light units of handledanimals (lower line) compared to non-handled animals (upper line) asdetermined by chemiluminescence measurement of superoxide production inwhole blood samples after challenge with PMA.

Example 5 illustrates application of the invention to determine if thestress of running a marathon causes a reduction in the ability of theimmune system to respond to challenge with reference to the followingfurther figures:

FIG. 4 shows typical data for a single marathon runner obtained usingblood samples taken pre- and post-marathon and determining the abilityof leucocytes in the blood samples to produce superoxide in response toPMA challenge (filled squares: pre-race control; open squares: responsein post-race sample).

FIG. 5(a) shows combined data for 16 marathon runners obtained usingblood samples taken pre- and post-marathon and determining the abilityof leucocytes in the blood samples to produce superoxide in response toPMA challenge (filled squares: pre-marathon control samples; opensquares: post-marathon samples).

FIG. 5(b) shows the non-PMA stimulated chemiluminescence measured at thesame time points for the same blood samples (filled squares:pre-marathon control samples; open squares: post-marathon samples).

Example 6 describes in more detail the study indicated above comparingperceived stress and leucocyte coping capacity in two groups ofpatients, elective patients and emergency (acute) patients pre- andpost-cardio-pulmonary bypass surgery. The data for leucocyte copingcapacity at 35 minutes post-challenge, obtained using whole bloodsamples and chemiluminescence measurement, is presented in box plots inFIGS. 6 and 7 wherein phagocytic capacity per cell per min representsmeasured relative light units divided by the white cell count and thenumber of minutes post challenge with adjustment for backgroundactivity.

DETAILED DESCRIPTION OF THE INVENTION

A method of the invention may be a carried out on neutrophils obtainedfrom an individual in any manner whereby the neutrophils are in a samplesuitable for chemical-induced superoxide production but will mostpreferably for convenience be applied to neutrophils in a whole bloodsample. For example, in the case of a human, such a sample may beobtained by a simple finger prick. If need be, blood samples for use ina method of the invention may be treated with an anticoagulant. While awhole blood sample may be utilised directly, with or without ananticoagulant, it will be appreciated that a blood fraction comprisingneutrophils may alternatively be employed if desired. For example, asample comprising isolated leucocytes may be employed.

A sample to be tested, directly or after further processing, may beobtained during or shortly after exposure of the individual of concernto a suspected or known psychological stressor. Where one or moresamples is taken after exposure to a known or suspected psychologicalstressor, the initial such sample will generally be obtained as soon aspossible. Multiple samples from the same individual may be tested takenat different time points during and/or after exposure to a suspected orknown psychological stressor, e.g. within 1 to 5 minutes, 10, 15, 20 or30 minutes of exposure to the suspected or known psychological stressor.Multiple samples may be tested which have been obtained at differenttime points after exposure to a psychological stressor in order todetermine peaking of the stress response and/or the individual'srecovery rate. Alternatively, for simplicity and speed, an individual'scoping capacity for a known stress factor may be tested at a single timepoint after exposure to the stress factor. When testing for stress inanimals, the sample may be obtained whilst an animal is underanaesthesia.

A sample to be tested in accordance with the invention may be contactedwith any chemical inducer which is capable of stimulating superoxideproduction in neutrophils. The inducer used in the method may bepreferably phorbol myristate acetate (PMA), more particularly, forexample, the microbial product phorbol 12-myristate 13-acetateobtainable form Sigma. However, alternative inducers which might beemployed are well-known. They include N-Formyl-Met-Leu-Phe (fLMPchemotactic peptide), zymosan, lipopolysaccharide or adrenaline.Suitable lipopolysaccharide is obtainable from the cell wall of a gramnegative bacterium, for example E. coli. A combination of inducers maybe employed, e.g. a combination of PMA and fLMP. A suitableconcentration of inducer and period and temperature of induction may bedetermined if need be by simple experimentation. In the case of PMA,typically it may be added to a whole blood sample at about 10⁻⁶ M to10⁻³ M and the temperature of the sample maintained at about 37° C.Superoxide production may be measured at various time points or asuitable single measurement time point may be determined.

The production of superoxide in response to the inducer may be measuredby any known means. Preferably, however, chemiluminescent measurementwill be employed for convenience coupled with high sensitivity. For thispurpose, a suitable amplifier such as luminol(5-amino-2,3-dihydrophthalzine) or isoluminol(6-amino-2,3-dihydro-1,4-phthalazinedione) may be utilised to detectsuperoxide as described by Hu et al., Cell Signal (1999) 11, 355-360.Chemiluminesence may be conveniently measured using a portablechemiluminometer. In this way, coping capacity for stress factors may bereadily quantified even outside of a laboratory. As indicated above, thelevel of superoxide production may be measured at one time point afterthe addition of inducer to the sample, or at multiple time points.Typically, superoxide production is measured at multiple time points atintervals of between 1 and 10 minutes, for example every 5 minutes,after addition of inducer, over a total time period of between 5 minutesand 1 hour, for example over 30 minutes. Preferably, the level ofsuperoxide production is measured after a time period when controlneutrophils of the same species, which are free or substantially free ofstress-induced activation, will exhibit maximal superoxide productionunder the same in vitro conditions. This time point can be determined bytesting samples obtained from individuals who have not been exposed to apsychological stressor, and measuring superoxide production at varioustime points after addition of inducer. At the preferred time point, itwould be expected that the difference between superoxide production in asample from an individual exposed to stress, and one who has not beenexposed to stress, would be at its greatest. Therefore the test isideally more sensitive to any changes as a result of stress.

The production of superoxide in response to the inducer is compared tothe basal superoxide production in the absence of inducer of the samesample, or a further sample taken from the same individual. As indicatedabove, measured superoxide production in samples may also be divided bythe white cell count or neutrophil count to allow for differences incirculating leucocytes in the donors.

A method of the invention may be used to assess individual responses tothe same psychological stressor and compare coping capacities betweenindividuals for a particular psychological stressor. For example, two ormore individuals, e.g. two or more humans, may be exposed to a knownstressor, and their coping capacity measured at the same time point inaccordance with the preferred embodiment of the invention set out above.The results can then be compared to find which of the tested individualshas coped the best with exposure to the psychological stressor. Anindividual with a lower coping capacity as determined by in vitroinduced superoxide stimulation in neutrophils is less able to cope withthe stressor than an individual with a higher coping capacity determinedin the same manner after exposure to the same stressor. This methodwould therefore be of particular use in situations where, after astressful event, a rapid assessment of the individual's ability to copeis required or where it is desired to select individuals on basis ofbetter ability to cope than others with a recognised psychologicalstressor. Such a method could, for example, form part of a job selectionprocedure for human individuals where the job is known to entailexposure to psychological stress factors.

A method of the invention may also be used to compare the effects ofdifferent stressors on the same individual or group of individuals, forexample a sample of individuals from the same species. In this case, theindividual's coping capacity in response to each stressor is quantifiedand compared, in order to determine which is the most stressful.Alternatively, a putative stressor may be tested to determine if itresults in a change in the individual's physiological status which isindicative of stress.

As indicated above, methods of the invention are applicable not only tohumans but also non-human mammals and additionally birds. For example, amethod of the invention can be applied to farmed animals, such ascattle, pigs, sheep, lambs and poultry, e.g. chickens. Importantly, sucha method may be used to assess if farmed animals are suffering fromsignificant stress before slaughter with a view to providing meatproducts which might be labelled “stress-free”. A method of theinvention may also be applied to farmed animals with a view to providinga wide range of other products which might be labelled “stress-free” or“obtained from stress-free animals”, for example, dairy products such asmilk, dried milk, cheese, evaporated milk, condensed milks andice-cream, wool from sheep and lambs tested in accordance with theinvention, fine animal hair from animals such as alpacas, llamas,camels, yaks and goats tested in accordance with the invention andcoarse animal hair for brush-making. A method of the invention may beapplied to chickens for the purpose of obtaining eggs which might bemarketed as coming from “stress-free chickens”. Methods of the inventionmay also be applied to farmed animals to improve animal husbandrytechniques. For example, such methodology might be applied forcertification of farms as “stress-free farms”. Methods of the inventionalso find application in wildlife conservation and care as illustratedby the badger study already mentioned above and further described inExample 1 below.

Methods of the invention may be applied in assessing and improving theergonomic efficiency of a human working environment. Thus, methods ofthe invention may have application in office design, furniture design,lighting, colouring and heating of a working environment and design ofoffice equipment such as computers. Methods of the invention mayadditionally find application in such diverse fields as transportdesign, design of leisure facilities, e.g. spas and gyms, and shoppingfacilities to enhance well-being and reduction of stress-factors. Theymay also find application for certification of animal boarding houses,e.g. for cats and/or dogs, as “stress-free”.

It is also envisaged that assessment of coping capacity in accordancewith the invention will have many applications in the field of sportsscience. Example 5 describes using the methodology of the invention toconfirm the expectation that the stress of marathon running reduces theability of the immune system to respond to challenge as evidenced bydecreased leucocyte coping capacity in blood samples taken from marathonrunners post-marathon. Similar studies applied to sportsmen andsportwomen taking part in other sporting activities may be of value inimproving their performance and well-being.

The invention may also be applied in the field of medicine to assessstress of patients ahead of medical treatment, e.g. surgery, to assessstress associated with medical procedures and in optimising suchprocedures to minimise stress to patients which may aid recovery andreduce risk of further infection.

In a further aspect, the invention also provides a method of screeningfor a stress-relieving drug, which comprises administering a testcompound to an individual, exposing the individual to a psychologicalstressor, and measuring their coping capacity using a method accordingto the invention, and comparing their coping capacity afteradministration of the test compound to their coping capacity in theabsence of the test compound, wherein an increase in coping capacityafter administration of the test compound is indicative ofstress-relieving ability of said test compound.

The individual used in the above screening method may be a human oranimal, for example a mouse, rat, hamster, guinea pig or other mammal.The individual is subjected to a stressor, which may be for example anunpleasant stimulus or stressful situation. In humans, the stressor maybe completion of a task under time pressure, for example Raven'sprogressive matrices, or observing a stressful event which may befictitious or real. In animals, the stressor may be exposure to anunfamiliar environment. For example, a rat may be placed in anunfamiliar environment such as the “elevated cross”. The stressor mayalso be social stress, for example stress may be induced in mice byhousing them in individual cages and then introducing a stranger. Othersuitable psychological stressors may be identified using a method of theinvention for determining exposure to such a stressor.

Suitable test substances which can be tested in the above screeningmethod include combinatorial libraries, defined chemical entities andcompounds, peptide and peptide mimetics, oligonucleotides and naturalproduct libraries, such as phage display libraries and antibodyproducts. Typically, organic molecules will be screened, preferablysmall organic molecules which have a molecular weight of from 50 to 2500daltons. Candidate products can be biomolecules including, saccharides,fatty acids, steroids, purines, pyrimidines, derivatives, structuralanalogs or combinations thereof. Candidate agents are obtained from awide variety of sources including libraries of synthetic or naturalcompounds. Known pharmacological agents may be subjected to directed orrandom chemical modifications, such as acylation, alkylation,estelification, or amidification to produce structural analogs.

A stress-relieving drug identified by a screening method as above may besynthesized and/or formulated into a pharmaceutical composition.Formulation with pharmaceutically acceptable carriers and/or excipientsmay be carried out using routine methods in the pharmaceutical art. Thusthe manner of formulation will depend upon factors such as the nature ofthe substance and the condition to be treated. Any such substance may beadministered in a variety of dosage forms. It may be administered, forexample, orally (e.g. as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules), parenterally,subcutaneously, intravenously, intramuscularly, intrasternally,transdermally or by infusion techniques.

Once an individual has been diagnosed as suffering stress by a method ofthe invention, they may be provided with stress-relieving treatment, forexample, they may be administered a known stress-relieving drug. Thus ina still further aspect, the present invention provides use of astress-relieving drug in the manufacture of a medicament for treating anindividual, e.g. a human, who has been identified as suffering stress bya method of the invention. There is additionally provided a method oftreating an individual for stress, e.g. by administration of astress-relieving drug wherein said individual has been identified assuffering from stress by a method of the invention.

A stress-relieving drug for use in such a method may be one that reducesstress, improves or ameliorates the symptoms of stress, or a drug thattreats a stress-related disorder. For example, the drug may be a knownanxiolytic or antidepressant or an adjunct to such drug therapy, e.g.drugs such as tryptophan The main classes of anxiolytic drugs arebenzodiazepines, for example diazepam and alprazolam; 5-HT_(1A)-receptoragonists, such as bupirone; and β-adrenoceptor antagonists, for examplepropranolol. The main classes of antidepressant drug are tricyclicantidepressants, for example imipramine and amitriptyline; selectiveserotonin re-uptake inhibitors, for example fluoxetine, fluvoxamine,paroxetine and sertraline; monoamine oxidase inhibitors, such asphenelzine, tranylcypromine, clorgyline and moclobemide; and atypicalantidepressants, for example nomifensine, maprotiline, mianserin,bupropion and trazodone.

In a still further aspect, the present invention provides a method oftesting the efficacy of a proposed stress-relieving treatment whichcomprises exposing an individual, e.g. a human, to a psychologicalstressor in the presence and absence of said treatment and determiningtheir coping capacity as described above. The treatment to be tested maybe, for example, handling of a device, gadget or toy designed with theaim of bringing stress relief or exposure to a particular environment,e.g. a smell as in aromatherapy, a pattern of light and/or imagery orsound composed with a view to bringing stress relief. Efficacy of anysuch treatment will be indicated by an increase in challenge-inducedsuperoxide production indicative of an increase in coping capacity.

As indicated above, where it is desired to compare two regimes asstressors, e.g. two protocols for medical treatment or a known stressfulregime plus and minus a proposed means of alleviating stress, then itmay be chosen to directly compare coping capacity determined in testsamples taken from individuals before, during and after subjection tothe two regimes of concern after correction for the number of whitecells or neutrophils in each sample.

In a further aspect of the invention, there is provided a device forcarrying out a method of quantifying stress according to the inventioncomprising a portable chemiluminometer together with other components toenable a single device system for quantifying stress. A device of theinvention may desirably comprise a portable chemiluminometer linked to asystem for analysing the results to provide a stress measurement foreach sample.

The following examples illustrate application of the invention to bothwild animals and human volunteers.

EXAMPLES Example 1 Trapping and Transport of Wild Badgers

As indicated above, this study was carried out to test the predictionthat transport of badgers causes substantial additional psychologicalstress compared to trapping of such animals without transport asreflected in lower coping capacity determined from residual capacity ofblood neutrophils for in vitro activation.

METHODS

(a) Trapping and Transporting Badgers

Badgers were trapped in Wytham Woods, Oxfordshire, U.K (for details ofthe study site and its badger population see Macdonald & Newman (2002)J. Zool. 256, 121-138) in cage traps baited with peanuts in August andNovember 2001. Badgers were selected because a measure of stress forthis species would be immediately useful in studies of its unusualsocial system (Macdonald & Newman 2002) and its role in the epidemiologyof bovine TB. Also, studies, conservation and control of badgersnecessitate their capture and handling, so a means of evaluatingalternative procedures would be helpful. The particular population wasselected because their individual life histories have been monitored for14 years during which the utilised trapping and handling procedures wererefined to the highest welfare standards. Badger traps were set adjacentto badger setts between 14.00 and 17.00 h. Traps were checked eachmorning between 06.30 and 07.00 h and trapped badgers were transferredto individual holding cages.

Animals were then assigned to one of two experimental regimes: samplingat the site of capture, without transport (non-transported, n=8), orsampling immediately after transport (transported, n=8). Transportconsisted of a short ride of less than 10 minutes on a trailer pulled byan all-terrain quad bike. While in holding cages, badgers were coveredwith a cloth. Badgers were anaesthetised, either at their site ofcapture or after transport using an intra-muscular injection of ketaminehydrochloride (“Ketaset,” Fort Dodge, U.S.A.) at a dose of 0.2 ml/kg.Processing consisted of measurements of body weight and length, andrecording the sex and condition of the badger. Other measurements werealso taken as part of the ongoing badger population study. Blood wasremoved by needle venepuncture of the jugular vein, collected into atube containing the anticoagulant potassium EDTA (BD Vucutainer Systems,Plymouth U.K.) and taken immediately for PMA challenge. Differentialcell counts were made from blood smears fixed in alcohol and stainedwith May-Grunwald and Giemsa stains (minimum of 100 cells per slide).

(b) PMA Challenge and Measurement of Coping Capacity

To measure the background blood chemiluminescence levels, 10 μl of wholeblood was transferred into a silicon anti-reflective tube (Lumivial, E G& G Berthold, Germany), to which 90 μl of 10⁻⁴M luminol(5-amino-2,3-dihydrophthalzine; Sigma A8511) diluted in phosphate bufferwas added. The tube was then shaken gently. To measure thechemiluminescence produced in response to challenge, a further tube wasprepared as above, but with the addition of 10 μl of the microbialproduct phorbol 12-myristate 13-acetate (PMA;Sigma P8139) at aconcentration of 10⁻⁶M. The PMA was dissolved in a small amount ofdimethyl sulphoxide (DMSO; Sigma D 5879) and then diluted to aconcentration of 10⁻⁶M in PBS. For each tube, chemiluminescence wasmeasured for 30 seconds every five minutes in a portablechemiluminometer (Junior LB 9509 E G & G Berthold, Germany) for a totalof 30 minutes. When not in the chemiluminometer, tubes were incubated at37° C.

Although a range of leucocytes can produce a respiratory burst,neutrophils are responsible for the majority of oxygen free radicalproduction and so leucocyte coping capacity (LCC) was also examined perquantity of 10⁹ neutrophils/l. This also provided a method of examiningLCC in relation to the potential effects of changes in the number ofcirculating neutrophils after stress.

(c) Statistical Analyses

To compare differences in coping capacity in badgers with and withouttransport, multivariate analysis of variance (MANOVA) was used, with theresponse at each time interval as dependent variables, and transportregime as a treatment variable. This procedure was carried out on SPSSfor Windows release 10.0.5. Data were log transformed prior to theanalysis to ensure the data met assumptions of multivariate normality(Tabachnick & Fidell (1996), Using Multivariate Statistics, ThirdEdition, New York: HarperCollins College Publishers). Wilcoxon SignedRanks Tests were used to compare an individual's coping capacity withits basal response.

(d) Animal Welfare Considerations

The badger population at Wytham Woods is under long-term scientificinvestigation, and badgers are trapped and transported regularlythroughout their lives. Examining transport stress is part of continuingrefinement of best practice. Work was carried out under English Naturelicence 1991537 and Home Office licence PPL 30/1826.

RESULTS

Basal superoxide production in and coping capacity in two groups ofanimals, transported (n=8) and non-transported (n=8), are shown inFIG. 1. Transport significantly reduced coping capacity in badgers(F_(7,8)=4.5, p<0.05). The difference in coping capacity betweentransported and non-transported individuals was greatest at 15 minutes(F_(1,14)=8,9, p<0.05; FIG. 1). However, two of the non-transportedindividuals were caught during a night when ambient temperaturesunexpectedly fell below freezing, and these individuals did not show atypical response for this group, and in both cases their coping capacitywas no greater than their basal response (Wilcoxon Signed Ranks Test,for both cases Z<−1.0, p>0.1). The transport regime had no effect onbasal superoxide production (F_(7,8)=2.5, p>0.05). However, to ensurethat there was no bias in the LCC results because of individualdifferences in unstimulated levels of chemiluminescence, we subtractedthe basal from the PMA-stimulated values for each animal(PMA-unstimulated).

Transport had an effect on the PMA-unstimulated values from 10 minutes(F_(1,14)=4.1; P=0.06) with the difference peaking at 15 minutes(F_(1,14)=9.4; P-0.008) and thus showed a pattern consistent with theLCC values described above.

There were differences in the number of leucocytes between the twogroups, with transported individuals having significantly lower numbersof leucocytes (Mann-Whitney U=11.0, p<0.05). In addition, leucocytecomposition also differed between the groups: transported animals had ahigher percentage of neutrophils (Mann-Whitney U=13.5, p=0.05), and acorrespondingly lower percentage of lymphocytes (Mann-Whitney U=7.5,p<0.01). Calculation of LCC per 10⁹ neutrophils 1⁻¹ gave the samepattern as overall LCC, indicating that activity per neutrophil wasgreater in non-transported individuals (see FIG. 2).

These results indicate that stress dramatically reduces the potential ofcirculating leucocytes to produce free radicals and support that copingcapacity is a quantifiable measure of the stress associated with aspecific event. The rather uniform depression of the coping response inall transported individuals suggests that this stressor over-rode thesources of individual variation that characterised the coping responsesof non-transported individuals.

Example 2 Quantification of Stress Induced by Handling of Wild SmallAnimals

The purpose of this study was to investigate the stress response tohandling in small wild animals, more particularly in wood mice (Apodemussylvaticus) and bank voles (Clethrionomys glareolus), by measuring theability of animals' circulating leucocytes to produce superoxide inresponse to PMA challenge.

METHODS

(a) Experimental Design

One group of animals was handled in the same way as for ecological orbehavioural studies and then anaesthetised. Handling brings about astress response typified by an increased heart rate and attempted escapebehaviour and handling is generally stressful for wild animals. Anothergroup of animals was anaesthetised without handling. Handled animalswere removed from their traps by gently tipping them into a plastic bag,and were then handled by their scruff, identified to species and placedin a holding pot and anaesthetised. Non-handled animals wereanaesthetised in their traps, without being removed and handled.Initially, the front door of the each trap was opened and the animalinside briefly observed to ensure it was in a suitable condition forundergoing anaesthesia. All traps were then left in a dark, quiet roomfor at least 30 minutes prior to the experiment to ensure the initialobservations did not interfere with the experiment. The non-handledanimals served to provide a comparative sample of animals that could beused to determine if handling causes supression of coping capacity. Allanimals were anaesthetised using a mixture of oxygen and isoflourane(Mathews et al., Vet. Rec. (2002) 150, 785-787).

The small mammals used in the study were trapped overnight in Longworthlive-traps placed in pairs at 5 m intervals along hedgerows on twocommercial dairy farms in South-West England. Traps were baited withapprox 14 g of ‘Josie’ rabbit food (Delta Pet Foods Ltd., Llandovery,Camarthenshire, UK), and apple (approx 5 g) as a source of food andwater. Laboratory trials revealed that this bait mix is readily consumedby both species. The amount of bait provided meant that food waseffectively available ad libitum during the period in the trap. Trapswere set at 1500 hours and collected at 0800 hours the next morning.

While under anaesthesia, the animal's sex and reproductive status (basedon testes status in males and indications of mating or pregnancy infemales) was determined. Only males and females were included in thestudy which were in breeding condition. Animals were then weighed to thenearest 0.1 g on a digital balance and their body length from snout tovent measured to the nearest 1 mm. Some juveniles were considered to betoo small for sampling, and as a result only adult animals (15 g andover) were included in the analyses. Animals were also given anindividual fur clip mark that could be used to later identify them ifthey were recaptured. As an index of the resources available to theanimal, body weight was used. This was appropriate because, for smallmammals, body weight is more closely related to the level of storedreserves than calculated body condition indices. We also recorded thepresence or absence of fleas, since the presence of these relativelycommon parasites could potentially influence the animal's energybalance. Work was carried out under Home Office Licence PPL 30/1826.TABLE 1 Details of the forty animals used in the study. Wood Mice Bankvoles Handled 9 16 Not handled 6  9 Weight range, mean (SD) 23.0 (3.1)22.6 (2.3) Fleas: present 7  8 absent 8 17

(b) PMA Challenge and Measurement of Coping Capacity

Blood was removed from the animals by tail venepuncture and 30 μl wascollected from each animal in a heparinised capillary tube. To measurethe unstimulated blood chemiluminescence levels, 10 μl of whole bloodwas transferred into a silicon anti-reflective tube (Lumivial, E G & GBerthold, Germany), to which 90 μl of 10⁻⁴M luminol(5-amino-2,3-dihydrophthalzine; Sigma A8511) diluted in phosphate bufferwas added. The tube was then shaken gently. To measure chemiluminescenceproduced in response to challenge, a further tube was prepared as above,but with the addition of 10 μl phorbol 12-myristate 13-acetate (PMA;Sigma P8139) at a concentration of 10⁻⁴ M. The PMA was dissolved in asmall amount of dimethyl sulphoxide (DMSO; Sigma D 5879) and thendiluted to a concentration of 10⁻⁴ M in PBS. For each tube,chemiluminescence was measured every five minutes in a portablechemiluminometer (Junior LB 9509 E G & G Berthold Germany) for a totalof 30 minutes. When not in the chemiluminometer, tubes were incubated at37° C. Leukocyte coping capacity (LCC) was calculated for eachindividual as the whole blood chemiluminescence measured in response toPMA challenge minus the chemiluminescence measured in unstimulated wholeblood.

(c) Statistical Analyses

To compare differences in coping capacity with and without handling, amultivariate GLM (General Linear Model) was used. The chemiluminescencelevels at each time interval were the dependent variables. This approachwas chosen because it allowed examination of the effect of the treatmentat each time interval, giving more detail on the nature of the effectsof the treatment and permitted identification of the time interval atwhich the treatment effect was greatest. Univariate GLM was also used toexamine the factors which significantly affected peak coping capacity.The coping capacity values were log transformed prior to the analyses tomeet the GLM requirement of normality of data. A small number ofnegative values (no smaller than −10) were treated as zeros. Thisprocedure was carried out on SPSS for Windows release 10.0.5.

RESULTS

Forty animals were caught for this study and of these 25 were handled(16 bank voles and 9 wood mice) and 15 were not handled (9 bank volesand 6 wood mice) prior to anaesthesia. Handling brought about a markedreduction in LCC, and reduced the variation in LCC (see FIG. 3). Amultivariate GLM revealed that handling significantly reduced LCC(F_(6,33)=5.1, P=0.001) and this was the case at all time intervals (5minutes F_(1,38)=9.9, P=0.003; 10 minutes F_(1,38)=20.7, P=0.001; 15minutes F_(1,38)=26.3, P=0.001; 20 minutes F_(1,38)=30.4, P=0.001; 25minutes F_(1,38)=24.9, P=0.001; 30 minutes F_(1,38)=22.7, P=0.001).There was no effect of species in the model, either as a variable on itsown (F_(6,31)=0.3, P=0.9) or as an interacting variable with handlingtreatment (F_(6,31)=0.4, P=0.9).

LCC was most variable for handled and non-handled animals after 25minutes, which was also the peak of LCC. Even given the more uniformresponse of handled animals, the range of LCC in these individuals after25 minutes was 220 relative light units (minimum −10, maximum 210, mean39.3, SD 62.9). AGLM was created using the LCC data at 25 minutes(LCC₂₅), which included treatment, treatment×body weight interaction andtreatment×flea interaction. This model indicated that at LCC₂₅ there wasa significant treatment×body weight interaction (F_(2,35)=4.1, P=0.024),but no evidence of a significant treatment×flea interaction(F_(2,35)=0.34, P=0.91). To explore the treatment×body weightinteraction further we created individual GLMs for handled andnon-handled animals. These models indicated that body weight wassignificantly and positively associated with LCC₂₅ in non-handledanimals (FIG. 2; F_(1,13)=5.6, P=0.034), but this relationship was lostwhen animals were handled (F_(1,23)=0.6, P=0.42).

This study thus again indicates that leucocyte coping capacity is aquantifiable measure of stress. Handling, a recognised stressor,suppressed the response of circulating leukocytes to PMA. There was noevidence that the level of suppression brought about by handling wasrelated to the animal's body weight, although heavier non-handledanimals had a greater peak LCC value. There was variation in theresponse of handled animals to PMA, albeit much reduced in comparisonwith the response of animals that had not been handled, but no evidencewas found to support that the level of resources available to individualanimals could explain this variation.

Example 3 Observing a Fictitious Stressful Event

The purpose of this study was to assess the effect of watching apotentially stressful event on human volunteers.

METHODS

(a) Subjects

Local ethical committee approval from Coventry University EthicsCommittee and informed consent was obtained before this study commenced,in accordance with the declaration of Helsinlki. 14 subjects allmoderately fit and healthy aged between 20-26 participated in the study.Exclusion criteria included suffering from psychiatric illness,respiratory or cardiovascular disease, smokers, or prescription medicinetaken within the previous month.

(b) Design

The experiments were performed in the afternoon according to highlystandardised procedures. Subjects were instructed to avoid exercise oralcohol for 48 hours before the study and be fasting for 2 hours beforethe study began. Both groups sat quietly for 15 minutes to obtainresting blood pressure, heart rate and resting blood samples. Bloodsamples were then taken from all subjects 15 minutes before beingexposed to either no stress (control conditions) or psychological stressin the in the form of exposure to the horror film. This time point wasdesignated −15 minutes. Subjects had no prior knowledge as to whichgroup they would be assigned to.

8 subjects were exposed to psychological stress in the form of an83-minuite horror film that none of the subjects had previously seen(The Texas Chainsaw Massacre, 1974, directed and produced by TobeHooper, Everett Collection, Inc). The remaining 6 subjects acted as acontrol group. They were instructed to sit quietly for the same lengthof time (83 minutes), under similar lighting conditions and were givenemotionally non-stimulating material to read if they so wished. Thisconsisted of text books, articles from journals and health informationleaflets. A repeated measures design was implemented with two groups:the experimental group (watching the horror film) and a control group.Blood samples, heart rate and blood pressure measurements were taken 15minutes before, and just after (85 minutes) being exposed to either the‘stressful event’ or to control conditions.

(c) Heart Route & Blood Pressure Measurements

A heart rate transceiver (Polar, Heart rate monitor) was attacheddirectly to the chest and heart rate was monitored. Participants wereseated, asked to make themselves comfortable, close their eyes andbreathe orthonasally. This procedure was carried out for a period offifteen minutes, in order to minimise possible stress levels experiencedprior to, or upon arrival, at the laboratory. At the end of this periodbaseline heart rate was recorded and the first blood sample taken (seesample protocol below). Participants were then instructed to either readquietly or in a separate room to watch the horror film. Upon completionof the task heart rate was recorded again, and further blood samplestaken. Systemic blood pressure (BP) was measured using an aneroidsphygmomanometer (Accoson, (Surgical) Ltd, London) and stethoscope(Harvard Ltd, Edenbridge, UK). Finger stick samples of blood 20 μl wereobtained at the specified time points.

(d) PMA Challenge and Measurement of Coping Capacity

To measure the background blood chemiluminescence levels, 20 μl of wholeblood was transferred into a silicon anti-reflective tube (Lumivial E G& G Berthold Germany), to which 90 μl of 10⁻⁴M luminol(5-amino-2,3-dihydrophthalzine; Sigma A8511) was added. The tube wasthen shaken gently. To measure chemiluminescence produced in response tobacterial challenge, 20 μl of phorbol 12-myristate 13-acetate (SigmaP8139) at a concentration of 10⁻³M was added. For each tube,chemiluminescence was measured for 30 seconds every five minutes in aportable chemiluminometer (Junior LB 9509 E G & G Berthold Germany) fora total of 30 minutes. When not in the chemiluminometer, tubes wereincubated at 37° C. The maximum response to PMA was noted.

(e) Data Analyses

Data are expressed as means±standard deviation from mean. A 2-tailedunpaired t-test was subsequently used to compare the 2 groups atbaseline and 85 minutes.

RESULTS

Table 2 shows the differences in the response to PMA between the horrorfilm group (n=8) and the control group (n=6). A highly statisticallysignificant (p<0.05) decrease was found at 85 minutes in the horror filmgroup when compared to the control group. The average heart rate changeof +14 for the horror film group represents a 20.6% increase. Theirsystolic and diastolic blood pressure rose by 10% and 16% respectively.The control group showed no such changes. TABLE 2 Summary of maximumresponse to PMA. −15 minutes Max response to PMA (RLU) Baseline 85minutes Control 320 (40) 340 (35)  Horror group 325 (27) 120 (60)*SD in brackets.*P < 0.05 from control.RLU = Relative Light Units

Watching a horror movie elicits a psycho physiological arousal which iscomparable to Canon's fear flight fight defence reaction, the so called‘stress response’ which involves stimulation of the hypothalamus, achange in peripheral resistance and an increase in the release of stresshormones including catecholamines and cortisol. This study also showsthat stress affected the responsiveness of a peripheral sample of blood.This can therefore be used as an indictor of stress.

An altered responsiveness to PMA indicates in vivo changes to the stateof leukocyte activation. In a state of stress, it is possible that theleukocytes actually release the contents of their granules (and are thusunable to respond to PMA) having already released the contents of theirgranules. Such leukocytes would be unable to respond to opportunisticinfections thus rendering the host more susceptible to disease, as wellas potential tissue damage from a host of proteolytic enzymes and oxygenfree radicals.

Example 4 Stressor Task METHOD

(a) Subjects

Local ethical committee approval from Coventry University EthicsCommittee and informed consent was obtained before this study commenced,in accordance with the declaration of Helsinki. 12 undergraduatestudents (aged 18-23 years) were assigned to either an experimentalgroup (given test) or asked to sit quietly for an equivalent timeperiod. Exclusion criteria included suffering from psychiatric illness,respiratory or cardiovascular disease, smokers, or prescription medicinetaken within the previous month.

(b) Stressor Task

The test was a written visual spatial task test comprising 36 questions,on which a 15 minute constraint was imposed (Ravens J. C. AdvancedProgressive Matrices, set II, Oxford Psychologists Press, Oxford, 1994).A small finger stick blood sample (Boebringer Mannheim, Soft Clix) wastaken before and immediately after task completion. 20 μl of blood wascollected in a pipette.

(e) PMA Challenge and Measurement of Coping Capacity

To measure the background blood chemiluminescence levels, 20 μl of wholeblood was transferred into a silicon anti-reflective tube (Lumivial E G& G Berthold Germany), to which 90 82 l of 10⁻⁴M luminol(5-amino-2,3-dihydrophthalzine; Sigma A8511) was added. The tube wasthen shaken gently. To measure chemiluminescence, 20 μl of phorbol12-myristate 13-acetate (Sigma P8139) at a concentration of 10⁻³M wasadded. For each tube, chemiluminescence was measured for 30 secondsevery five minutes in a portable chemiluminometer (Junior LB 9509 E G &G Berthold Germany) for a total of 30 minutes. When not in thechemiluminometer, tubes were incubated at 37° C. The maximum response toPMA was noted.

(d) Data Analyses

Data are expressed as means±standard deviation from mean. A 2-tailedunpaired t-test was subsequently used to compare the 2 groups atbaseline and 15 minutes.

RESULTS

Table 3 shows the effect of completing a stressor task on leukocyteresponsiveness to PMA. The experimental group had significantly reducedresponses to PMA (p<0.05, unpaired t test) compared to controls.Therefore completion of a stressor task diminished responsiveness toPMA. TABLE 3 Summary of maximum response to PMA. Max response to PMA(RLU) Baseline 15 minutes Control 420 (40) 380 (25)  Experimental Group380 (30) 160 (70)*Mean +/− Standard deviation (SD) in brackets.*P < 0.05 from control.RLU = Relative Light Units

Example 5 Determination of the Effect of Marathon Running on the Abilityof Blood Neutrophils to Respond to PMA Challenge in Vitro

Study Design

Sixteen marathon runners were included in the study who during thecourse of the study undertook the London marathon. Blood samples weretaken from the runners the day before the race and immediately followingthe race. For each blood sample, basal chemiluminescence andchemiluminescence with PMA-challenge was determined in similar manner asdescribed in previous examples. Chemiluminesence measurements were madeat 5 minute intervals up to 35 minutes.

RESULTS

FIG. 4 shows typical data for a single runner. On the day before themarathon, the runner showed response to PMA-challenge as demonstrated bythe spike in relative light units (RLU) in the pre-race response line.In contrast, the post-race response line shows no such spike.

Most pre-race blood samples showed a peak response to PMA-challengeafter 15 to 20 minutes (see FIG. 5(a)). The magnitude of the peakresponse was highly variable. In contrast, the data for post-racesamples indicated strong damping of PMA-induced superoxide productioncompared to the control pre-race samples. The non-PMA stimulated resultsfor the same samples are shown in FIG. 5(b). This data is in keepingwith the well-known phenomenon that elite athletes are more at risk fromcertain types of infection than other people and suggests utility of theinvention across a wide range of sporting activities for betterunderstanding of stress in sportsmen and sportswomen and in findingmeans to ameliorate the effects of stress or reduce stress in suchpeople.

Example 6 Evaluation of Patients' Psychological Perceptions andImmunological Responses Pre and Post Cardio-Pulmonary Bypass Surgery(CPB) in two Groups of Patients a) Those Who Have Waited for ElectiveSurgery b) Patients Who Are Admitted on an Emergency (Acute) BasisMETHODS

(a) Study Design

Ninety patients who were to undergo CPB were assigned to one of twogroups. An ‘elective’ group n=64 (51 males, 13 females) who had been onwaiting list and an ‘acute’ group n=26 (22 males, 4 females). Patientswere tested on the evening before surgery and at clinic six or sevenweeks after discharge using standard measures to assess psychologicalwell-being. Small peripheral blood samples were taken. Neutrophils'‘Current’ activity was determined from whole bloods' capacity to reducenitro-blue tetrazolium (NBT) (% NBT positive cells), and leukocytecoping capacity, from stimulated whole blood, luminol-dependantchemilunlinescence.

Research was approved by the local health authority ethics committee(University Hospitals Coventry and Warwickshire, NHS Trust), theWalsgrave Hospital Research and Development Committee the School ofScience and the Environment ethics committee (Coventry University) andCardiothorasic consultant Walsgrave hospital, whose patients formed thebasis of this research.

The 90 adults in the study underwent cardiopulmonary bypass surgery foreither coronary artery bypass grafting (CABG), mitral valve (MV) surgeryor aortic valve (AV) surgery and were recruited on the night prior tosurgery. The inclusion criteria were:

-   -   Ability to communicate in both spoken and written English        language;    -   Informed consent;    -   The patient was not involved in any other research or        experimental procedures.

The exclusion criteria were:

-   -   a Previous coronary bypass grafting;    -   An immune deficiency disorder;    -   An inflammatory condition not related to the heart problem.

The males of the elected group ranged in age from 38 to 80 (mean 63.61,SD 8.75) and the females of the same group ranged in age from 38 to 81(mean 64.86, SD 13.29). In the acute patient group, the 22 males wereaged between 24 and 80 years (mean 64.86, SD 13.29) and the 4 femaleswere aged between 60 and 81 years (mean 71.75, SD 8.85).

Table 4 below shows a comparison of the patient profiles of the twogroups observed. The elective group was reduced to 62 patientspost-surgery due to death of one patient and one patient still being inhospital with neurological complications. The acute group was reduced to20 post-surgery due to the death of 5 patients in hospital and onepatient opting out of surgery. TABLE 4 Preoperative comparative patientprofile of both groups. Elective Group Acute Group Males Females MalesFemales Number 51 13 22 4 Age Range 38-80 38-81 24-80 60-81 Mean (SD)63.61 66.62 64.86 71.75 (8.75) (10.67) (13.29) (8.85) Type of SurgeryCABG 37  4 12  3 Valve 10  8 6 1 CABG & Valve 3 1 4 CABG, Valve & other1 Angina Status Asymptomatic 6 4 6 NYHA 1 7 2 NYHA 2 14  2 1 NYHA 3 20 3 3 1 NYHA 4 4 2 12  3 Dyspnoea Status Asymptomatic 7 1 NYHA 1 11  2NYHA 2 14  2 4 NYHA 3 11  4 4 2 NYHA 4 8 7 11  2 Coronary Disease 1Vessel 3 2 Vessel 9 2 1 3 Vessel 30  3 15  2 N/A Valve only 9 8 6 2 ASAGrade Healthy 3 Mild 11  1 2 Severe 32  11  11  3 Severe/Life 5 1 9 1threatening Previous Infarct None 30  11  8 2 Single 15  2 10  2Multiple 6 4 Post Surgery Status Alive 49  13  18  2 Still hospitalized1 Deceased in Hospital 1 4 1 Opted out/surgery 1Key To Table 4CABG = Coronary Artery Bypass GraftingValve = Mitral or Aortic Valve replacement or repairThe physicians and the surgeons, on the following scale, preoperatively,graded both angina and dysnoea status:NYHA 1 = No limitations of normal physical activityNYHA 2 = Ordinary physical activity causes discomfort/dyspnoeaNYHA 3 = Moderate to great limitations of ordinary physical activityNYHA 4 = Unable to perform any physical activity withoutdiscomfort/dyspnoeaAs a gross predictor of overall outcome, post-operatively, ananaesthetist graded each patient on the evening before surgery, usingwhat is termed the ASA grade, as follows:ASA I = Healthy patientASA II = Mild systemic disease - no functional limitationASA III = Severe systemic disease - definite functional limitationASA IV = Severe systemic disease that is a constant threat to life

(b) Procedure

Patients were approached by the researcher upon their arrival athospital or on the evening prior to surgery (often one and the sametime). Briefly, patients were told that the research was to measuretheir levels of perceived stress and anxiety and the effects this mayhave on neutrophils before and after surgery. They were additionallytold that they would be required to complete a number of simplequestionnaires and to provide a small sample of blood from a finger (theequivalent of three drops) on two occasions, the first being during thatevening and the second at the post-operative clinic approximately sixweeks after surgery. Any questions were answered and an informationsheet was given to each patient and any relatives in attendance.

The researcher then gave the patients time to think about this,returning later with consent forms and asking again if they were willingto take part, explaining that they could if they wished withdraw at anytime. If consent was given, demographic data was recorded and patientswere asked to complete the questionnaires. A finger stick blood samplewas taken from a cleaned finger using a Soft-Clix™ and sterile lancetand a Gilson pipette was used to collect the blood (3 by 10 μl sampleswere taken). One sample was placed in 40 μl of luminol in a luminometertest tube. The second sample was mixed with an equal quantity ofnitro-blue tetrazolium (NBT).and the third sample was placed-in 90 μl ofdiluting fluid for white cell counting (see sample protocol below). Thewound was dressed if required. Any questions were answered and then thepatients were left in peace.

At the post-operative clinic approximately six to seven weeks aftersurgery, patients were again seen by the researcher and were asked ifthey wished to continue. If so, the questionnaires were again completedand blood samples taken again.

(c) Protocols

(i) Measurement of PMA-Challenge Induced Superoxide Production in BloodSamples

10 μl of whole blood was mixed with 40 μl of luminol diluted to 10⁻⁴Mwith phosphate buffered saline (PBS) in a luminometer test-tube. Thesample was placed in a luminometer and relative light units (RLU) wererecorded after a period of 60 seconds. This initial test was carried outto determine the background activity of the sample so that whencalculating coping capacity this background activity could be deducted.20 μl of phorbol-12-myristate-13-acetate (PMA) and 20 μl offormyl-Met-Leu-Phe (fMLP) were then added and RLU were recorded at theend of 5 minutes, 20 minutes and 35 minutes after stimulation. Copingcapacity was determined by dividing the RLU by the white cell count andthe number of minutes of the test and adjusting for background activityand presented as phagocytic capacity per cell per min.

(ii) Nitro Blue Tetrazolium Test (NBT)

This test used to determine the ‘current’ oxidative state of theneutrophils within the sample, as determined by the oxidative capacityof the neutrophils to reduce nitro-blue tetrazolium and form a formazanpigment.

10 μl of blood was mixed with 10 μl of NBT, diluted at 1 mg per ml, andincubated at 37° C. for ten minutes sample was then smeared onto twoclean microscope slides and air dried. Samples were then flooded withAccustain Wright stain, rinsed with distilled water and dried.Percentage of ‘activated’ neutrophils was determined by careful countingunder the light microscope.

(iii) Whole White Cell Count (WCC)

A manual WCC count was undertaken for each patient. 10 μl of blood wasmixed with 90 μl of 2% acetic acid coloured pale purple with crystalviolet and gently mixed to lyse the red blood cells. A sample was thenplaced on a haemocytometer and a careful count was made. A mean WCC perml was calculated from at least five fillings of the haemocytometer.

(iv) Measures

Each patient completed the ten-item Perceived Stress Scale (Cohen,Kamarck & Mermelstein, 1983), the 14-item Hospital Anxiety DepressionScale (Zigmond & Snaith) and the 20-adjective, Positive and NegativeEffect Scale (PANS) (Watson, Clark & Tellegen 1988). In addition, asix-item visual analogue scale was constructed to investigate theperception of stress specifically about the illness, the self-efficacyof the patient and their locus of control. The items from this scale arelisted below:

-   -   1. How ‘stressed’ do you feel about you health Right Now?    -   2. How much control do you feel you have over you health in        general?    -   3. How much control do you feel you have over you heart        condition Right Now?    -   4. Overall how confident are you that your heart condition will        have improved in 12 months time?    -   5. How much control do you feel health professionals have over        your heart condition Right Now?    -   6. How confident are you that you can do all the things        necessary to manage your condition and/or its treatments on a        regular basis?

Each item could be scored on a scale from 0 (no stress, no control, notconfident) to 10 (stress as bad as it can be, complete control,completely confident).

Clinical pre-operative data was also recorded for all patients includingprevious. infarct; angina status, lung function and grading by theanaesthetist (ASA grade) (data obtained from hospital record database).

RESULTS

(i) Summary

Results revealed a consistent, statistically significant associationbetween stress and % NBT cells pre and post-surgery (p<0.001) in bothgroups. There was a significant difference in perceived stress (p=0.009)between the groups, pre-surgery, both significantly reducedpost-surgery. No significant differences were found between or withinthe groups for anxiety, depression, and positive and negative effect.Interestingly, the ‘acute’ group had the highest perceived psychologicalstress as determined by self-administered questionnaires. However,coping capacity as determined from blood samples was significantlygreater in the ‘acute’ group both before and after surgery (p<0.02).

(ii) Perceived Stress, Anxiety and the Current Activity (NBT) ofNeutrophils.

(a) Pre-Surgery

A consistent and significant relationship between PSS and percentage ofNBT positive neutrophils was found in both groups (r=0.912, p=0.0001 inthe elective group and r=0.878, p=0.0001 in the acute group).

There were also found consistent and significant relationships in bothgroups between the percentages of NBT positive neutrophils and anxiety(r=0.670, & r=0.616, p=0.0001), depression (r=0.632, & r=0.664,p=0.0001), negative effect (r=0.601, p=0.0001 & r=0.581, p=0.001) anddyspnoea status (r=0.275, p=0.028 & r=0.399, p=0.044). There was alsofound a significant relationship between NBT and health related stresswithin the elective group (r=0.499, p=0.0001) but not in the acutegroup.

(b) Post Surgery

A consistent and significant relationship between PSS and percentage ofNBT positive neutrophils was found in both groups (r=0.851, p=0.0001 inthe elective group and r=0.944, p=0.0001 in the acute group.

Consistent and significant relationships were found between thepercentage of NBT positive neutrophils and PSS (r=0.851 & r=0.944,p=0.0001), anxiety (r=0.488, & 0.864, p=0.0001), depression (r =0.475,p=0.0001 & r=0.688, p=0.001), positive effect (r=−0.329, p=0.09 &r=−0.593, p=0.006), negative effect (r=0.494, & r=0.831, p=0.0001) andhealth related stress (r=0.456, & r=0.707, p=0.0001).

The above results, from both the pre and post surgery time points,suggest that the percentage of NBT positive neutrophils showconsiderable associations with the psychological state of the patientsstudied with the negative states (e.g. anxiety, depression, PSS,negative effect and health related stress) increasing as the percentageof NBT neutrophils increases. Positive effect shows a negativeassociation suggesting that as positive effect increases the percentageof NBT positive neutrophils decreases. However few or no associationsare seen between NBT and disease indicators.

(iii) Coping Capacity of Neutrophils

Analysis of the Chemiluminesence data (Mann-Whitney U test) comparingthe groups at each time-point revealed no significant differencesbetween the groups at 5 minutes or 20 minutes for pre- or post-surgery,samples (see Tables 5 and 6 below). However, a consistent andsignificant difference in coping capacity was found at 35 minutes forboth pre- (p=0.018) and post-surgery (p=0.019) (see FIGS. 6 and 7).TABLE 5 Comparison of the phagocytic capacity at the three time points(pre-surgery) Elective Group Acute Group n = 64 n = 26 Time median maxmin median max min p  5 mins. 0.00743 0.2112 −0.0011 0.00775 0.0194−0.0027 ns 20 mins. 0.01547 0.0967 0.0017 0.17423 0.0684 0.0026 ns 35mins. 0.35613 0.5069 0.0065 0.05610 0.1679 0.0153 0.018

TABLE 6 Comparison of the phagocytic capacity at the three time points(post-surgery) Elective Group Acute Group n = 62 n = 20 Time median maxmin median max min p  5 mins. 0.00191 0.0226 −0.0042 0.00599 0.0123−0.0011 ns 20 mins. 0.01701 0.0883 −0.0123 0.01954 0.1086 0.0028 ns 35mins. 0.03299 0.1677 −0.0163 0.04368 0.1137 0.0224 0.019

Bivariate correlations of the data show no significant associationsbetween coping capacity and either the psychological variables or thephysiological indicators.

(iv) Perceived Stress and Health Related Stress.

The level of PSS reported in both groups prior to surgery wassignificantly higher than post surgery (t=2.72, p=0.008 & t=3.44,p=0.001). It is also clear from the mean scores that the acute groupwere reporting significantly higher levels of PSS than the electivegroup (t=−2.674, p=0.009). This is also true of health related stresswhich was significantly higher pre-surgery than post-surgery in bothgroups (t=5.19, p=0.000, & t=3.76, p=0.001). Again, the highest meanscore is in the acute group pre surgery. There were no significantdifferences between the health related stress scores between the groupspre-surgery. The ‘norm’ PSS for this population was 12±6.3 (SD) comparedto slightly higher in the elective group (14.72,±7.21), but much higherin the acute group (19.12,±6.71), suggesting that the acute group wasthe more ‘stressed’. Post surgery scores were near to population‘norms’.

(v) Conclusion

Whilst patients from the acute group perceived themselves to be morestressed than patients from the elective group, their leucocytes had agreater coping capacity than those from the elective group. Inphysiological terms they would be less susceptible to opportunisticinfections and stress-related illness.

1. A method for determining whether an individual, which is a mammal orbird, is experiencing changed physiological status arising from exposureto a psychological stressor, the method comprising: (a) contacting atest sample comprising neutrophils obtained from said individual with aninducer capable of stimulating superoxide production in neutrophils,under conditions suitable for such stimulation; (b) determining theincrease in superoxide production above basal in said test sample aftera time period when neutrophils of the same species in a control sample,which are free or substantially free of stress-induced activation or atleast derived from one or more individuals exposed to the same regimeminus a factor to be tested as a psychological stressor, will exhibitsuperoxide production under the same in vitro conditions; and (c)comparing the increase in superoxide production above basal observed insaid sample with the increase in superoxide production above basalobserved in a control sample as defined in (b) above under the sameconditions; wherein lower superoxide production in said test sample isindicative of the effect of a psychological stressor on the individual'sphysiological status.
 2. A method according to claim 1 for determiningthe coping capacity of an individual for exposure to a psychologicalstressor, wherein prior to step (a) said individual is exposed to saidpsychological stressor for a time period whereby neutrophils in anindividual of the same species who is susceptible to stress induced bysaid stressor will exhibit increased superoxide production and whereinthe degree of further in vitro induced superoxide production in saidtest sample above basal determined in step (c) is a measure of copingcapacity.
 3. A method according to claim 1 wherein said sample comprisesisolated leucocytes
 4. A method according to claim 1 wherein said sampleis a whole blood sample.
 5. A method according to claim 1, wherein theindividual is human.
 6. A method according to claim 1 wherein theindividual is a bird, such as a chicken.
 7. A method according to claim1 wherein the individual is a farmed animal, such as a cow, pig, sheep,lamb or poultry.
 8. A method according to claim 1 wherein the individualis a wild mammal.
 9. A method according to claim 1, wherein the inducercapable of stimulating superoxide production in neutrophils is phorbolmyristate acetate (PMA), N-Formyl-Met-Leu-Phe (fLMP chemotacticpeptide), zymosan, lipopolysaccharide or adrenaline.
 10. A methodaccording to claim 1, wherein superoxide production is detected usingluminol or isoluminol as an amplifier and the resultingchemiluminescence is measured.
 11. A method according to claim 1,wherein the inducer capable of stimulating superoxide production inneutrophils is phorbol myristate acetate (PMA), superoxide production isdetected using luminol as an amplifier and the resultingchemiluminescence is measured.
 12. A method of screening for astress-relieving drug, the method comprising: (a) administering a testcompound to an individual; (b) exposing said individual to apsychological stressor and measuring their coping capacity using amethod according to claim 2; and (c) comparing their coping capacityafter administration of the test compound to their coping capacity inthe absence of the test compound, wherein an increase in coping capacityafter administration of the test compound is indicative ofstress-relieving ability of said test compound.
 13. A method accordingto claim 12, wherein the individual is a non-human mammal.
 14. A methodaccording to claim 12, further comprising synthesizing astress-relieving drug identified by said method, and/or formulating thedrug into a pharmaceutical composition.
 15. (canceled)
 16. A method oftreating an individual suffering from stress which comprises providing astress-relieving treatment, such as administering a stress-relievingdrug, to an individual identified as suffering from stress using amethod according to claim
 1. 17. A method of testing the efficacy of aproposed stress-relieving treatment which comprises exposing anindividual to a psychological stressor in the presence and absence ofsaid treatment and determining their coping capacity in accordance withclaim
 2. 18. A method as claimed in claim 1 wherein the control sampleis replaced by a test sample comprising neutrophils taken from a secondindividual, the test samples being taken from different individuals atthe same time point before, during, or after subjection to two differentregimes to be compared as stressors, and wherein the superoxideproduction above basal determined for each sample is adjusted to takeaccount of differences in white cell count or neutrophil count in thesamples.
 19. A method for comparing two regimes as stressors wherein amethod according to claim 18 is repeated at several time pointsemploying samples from more than one individual subjected to eachregime.
 20. A method as claimed in claim 18 or wherein the regimes to becompared are protocols for medical treatment.
 21. A method as claimed inclaim 18 wherein the regimes to be compared are a known stressful regimeplus and minus a proposed stress-alleviating treatment.
 22. A device fora carrying out a method according to claim 1 comprising a portablechemiluminometer and system for analysing the results to provide astress measurement.